Fluorescent polymeric sensor for the detection of creatinine

ABSTRACT

The present invention provides a creatinine sensor material having a first layer of a protonated pH sensitive fluorophore immobilized in a hydrophobic polymer, wherein the fluorophore can react quantitatively with ammonia and the transducing moiety of the fluorophore is neutrally charged when deprotonated; a second layer of creatinine deiminase and a polymer; and a third layer of a polymer. The present invention also provides a method for detecting creatinine using the creatinine sensor material and optical sensing devices that incorporate the creatinine sensor material.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to sensors for the detection ofcreatinine.

2. Description of Related Art

Continuous monitoring of creatinine can be accomplished by a number ofelectrochemical methods. Sensors utilizing such methods can be createdby immobilizing the enzyme creatinine deiminase onto the surface of anelectrode. The enzymatic hydrolysis of creatinine producesN-methylhydantoin and ammonia. At physiologic pH the ammonia isprotonated to form ammonium ions, which increase the electricalconductivity of the solution proximal to the electrode.

Creatinine can also be monitored using an optical sensor. The detectionof analytes by optical sensors usually requires the development offluorescent transducers which are specific for different analytes.Optical transducers can also be coupled to the detection of creatininevia the creatinine deiminase driven hydrolysis of creatinine, with theoptical transducer modulated by ammonium or ammonia.

Detection of ammonium requires an ammonium specific ionophore coupled toa chromophore that changes its absorption spectrum upon protonation, anda lipophilic anionic site. As such, sensors based on the detection ofammonium can be expensive and complex.

Detection of ammonia requires a protonated pH sensitive indicator(INDH⁺) which changes its absorption or fluorescence spectrum upondeprotonation:

    INDH.sup.+ +NH.sub.3 →IND+NH.sub.4.sup.+

There is also a drawback to designing a sensor based on detection ofammonia: namely the rapid protonation of ammonia at physiologic pH. ThepK_(a) of ammonium is 9.3, which is not a pH that supports maximumenzyme activity.

Hydrophobic polymers, optically transparent and permeable to the analyteof interest, are used with optical sensors when the analyte is a vaporor gas and is capable of diffusion into a hydrophobic membrane. Acomplication arises when hydrophobic polymers are used with certainfluorescent dyes. Sensors for ammonia require a protonated indicator.When combined with a hydrophobic membrane for the detection of ammonia,polyanionic pH indicators, which are the common variety of protonatedindicator and the type used in the fluorescent urea sensor described inRhines and Arnold (Anal. Chim. Acta, 231: 231-235 (1990)), do notproduce an activated and protonated fluorophore.

Sensors have been developed for the detection of creatinine based onenzymatic cleavage of creatinine. However, many of the sensors that havebeen developed for the detection of creatinine have been coupled to gaselectrodes (Thompson and Rechnitz, Anal. Chem., 46: 246-249 (1974);Kihara and Yasukawa, Anal. Chim. Acta, 183: 75-80 (1986)), unlike thepresent invention, which couples the enzymatic cleavage of creatinine todetection by a fluorescent polymer coating.

While various indicators for creatinine are known, many sensors exhibitproblems with interferences from pH and CO₂ effects, low sensitivity,slow response times and reversibility. From a manufacturing standpoint,it would therefore be desirable to develop an inexpensive sensor capableof detecting creatinine that has a high sensitivity, fast response time,and is reversible. It would also be advantageous for the sensor to beable to function in conjunction with sensors detecting other analytes.

SUMMARY OF THE INVENTION

The present invention provides a creatinine sensor material comprising afirst layer comprising a pH sensitive fluorophore immobilized in afirst, hydrophobic polymer, wherein the fluorophore can reactquantitatively with ammonia and the transducing moiety of thefluorophore is neutrally charged when deprotonated; a second layercomprising creatinine deiminase and a polymer; and a third layercomprising a polymer.

The present invention also provides a method for measuring creatininecomprising measuring the fluorescence of the creatinine sensor material;exposing the sensor material to a solution comprising creatinine;measuring the fluorescence change; and determining the concentration ofthe creatinine.

The present invention also provides an optical sensing device formeasuring ammonia concentration in a solution comprising creatinine,comprising a first layer comprising a pH sensitive fluorophoreimmobilized in a first, hydrophobic polymer, wherein the fluorophore canreact quantitatively with ammonia and the transducing moiety of thefluorophore is neutrally charged when deprotonated, on the surface of anoptical component which is transparent to incident and emissiveelectromagnetic waves; a second layer comprising creatinine deiminaseand a polymer on the surface of the first layer; and a third layercomprising a polymer on the surface of the second layer; wherein theoptical component is optically connected to means for collecting radiantemission to measure the fluorescence indicative of ammoniaconcentration.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows a creatinine sensor configuration as used in Example 1.

FIG. 2 shows the response of a creatinine sensor, in units offluorescence intensity, as a function of mg/dl creatinine concentration.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention relates to sensors for the detection ofcreatinine. The sensors of the present invention comprise a fluorophoreimmobilized in a hydrophobic polymer, wherein the fluorophore can reactquantitatively with ammonia and the transducing moiety is neutrallycharged when deprotonated.

The transducing moiety is the ring or group of rings in the molecularstructure of the pH sensitive fluorophore, which produces thefluorescence when radiated with the particular excitation energyrequired for excitation. This same segment of the molecule undergoes aresonance change due to protonation and deprotonation, and this changeresults in a change in the fluorescence which allows one to calibratethe fluorescence as a function of pH alteration. A substituent ring thatis not involved in the pH based resonance change may be negativelycharged when deprotonated; see e.g. the benzoic acid residue onrhodamine.

In the present invention, the detection of creatinine in a solutiondepends on the presence of the enzyme creatinine deirninase, whichcatalyzes the following reaction: ##STR1## The sensors of the presentinvention detect ammonia that is produced in the reaction above.Detection of ammonia is based on changes in the fluorescence spectrum ofthe fluorophore upon deprotonation, as shown in the reaction below:

    INDH.sup.+ +NH.sub.3 →IND+NH.sub.4.sup.+

The sensor material in its simplest form comprises one layer, that ofthe fluorescent polymer. In such a case, creatinine deiminase is addedto the solution in which creatinine is to be measured. In a preferredembodiment, the sensor material comprises three layers: a transducerlayer, an enzyme layer, and a protective layer. The transducer layer iscomprised of the fluorophore immobilized in a hydrophobic polymer. Theenzyme layer is comprised of the enzyme creatinine deiminase immobilizedin a polymer. The protective layer is another polymer.

Fluorophores suitable in the sensors of this invention are fluorescentpH indicators wherein the transducing moieties are neutrally chargedwhen deprotonated, and which exist in the protonated state in themicroenvironment of the polymer. Such fluorophores include acridineorange: ##STR2## and rhodamine dyes: ##STR3## wherein R¹ and R² areindependently an alkyl having between about 2 and 20 carbon atoms and R³is hydrogen or an alkyl having between about 2 and 20 carbon atoms.Acridine orange and rhodamine dyes are preferred fluorophores. Themodulation of the acridine fluorescence is generally measured with anexcitation at 489 nm and emission at 540 nm, but can be excited at otherwavelengths. A ratiometric readout can be achieved by exciting at twowavelengths and generating a ratio of the two emissions as a function ofammonia. The modulation of fluorescence of a rhodamine derivative,wherein R¹ and R² are both C₁₈ H₃₇, is generally measured with anexcitation at 530 nm and emission at 590 nm.

Hydrophobic polymers useful in the present invention for the transducermembrane are preferably polymers in which the fluorophore is at leastpartially soluble. Such polymers include but are not limited to:polystyrene, polyurethane, poly(ethyl cellulose), polydienes such aspoly(1,3-butadiene), butadiene-acrylonitrile copolymer,poly(dimethylbutadiene), and polyisoprene, polyalkenes such aspolyethylene, isobutane-isoprene copolymer, poly(4-methylpentene),polypropylene, polyethylmethacrylate, polytetrafluoroethylene,poly(vinyl alcohol), poly(vinyl chloride), and polyoxymethylene,cellulose and cellulose derivatives, such as cellulose hydrate,cellulose acetate, cellulose nitrate, ethyl cellulose, and celluloseethyl methacrylate, polymethacrylates such as poly(methyl methacrylate)and poly(ethyl methacrylate) as well as polysiloxanes, polyesters andpolycarbonates. A preferred polymer is ethyl cellulose. These polymersmay be used in both the transducing layer and the protective layer ofthe preferred embodiment.

The fluorophore and hydrophobic polymer are combined to form afluorescent polymer. The signal intensities of the fluorescent polymerare high as the dye is very soluble in the organic media of the polymer,and no quenching of fluorescence occurs (as in U.S. Pat. No. 5,506,148)due to the lack of negative charges on the dye molecule. Upon exposureto ammonia, the fluorescence of the fluorescent polymer decreases,consistent with the dye becoming deprotonated with the formation ofammonium ion in the fluorescent polymer. This sensor response isreversible when the source of ammonia is withdrawn, and the ammonia inthe sensor diffuses out of the membrane.

An onium compound can optionally be added to the fluorescent polymer.The onium compound adjusts the microenvironment pH of the fluorescentpolymer to enhance the sensitivity to ammonia. Onium compounds includeammonium, phosphonium and pyridinium compounds. Examples of suitableonium compounds include tetrabutylammonium hydroxide, tetrabutylammoniumchloride, cetyltrimethylammonium bromide, tetrabutylammonium hydrogensulfate, tetrabutylammonium trifluoromethane, tetrabutylammoniumacetate, tetraethylammonium bromide, tetraethylammoniump-toluenesulphoate, phenyltrimethylammonium chloride,benzyltrimethylammonium bromide, tetra-n-propylammonium bromide,benzyltriethylammonium tetrafluoroborate, n-dodecyltrimethylammoniumbromide, tetraphenylphosphonium chloride, n-hexadecylpyridinium bromide,triphenyl phosphonium chloride, tetrabutylphosphonium bromide andhexadecyltrimethylammonium hydroxide. Preferred onium compounds arequaternary ammonium compounds, such as tetrabutylammonium hydroxide.

The sensor material can be prepared by dissolving the fluorophore andpolymer in a suitable solvent, such as an alcohol, toluene,tetrahydrofuran or other organic solvent known in the art for dissolvingthe hydrophobic polymer. In general, the amount of fluorophore to beused should be between about 0.05% and 0.5% of the total mass. Thefluorophore is preferably uniformly distributed throughout the resultingfluorescent polymer.

A membrane or film can then be formed from the dissolved fluorescentpolymer by any suitable method known in the art, such as spincoating orbrushing onto a non-reactive substrate, such as glass, plastic orceramic. Alternatively, the fluorophore can be covalently attached tothe polymer, as described in U.S. Pat. No. 5,005,572.

The enzyme layer is comprised of the enzyme creatinine deiminasedissolved in a hydrophilic or hydrophobic polymer, which is thendeposited onto the transducer layer. The enzyme layer can be depositedonto the transducer layer in a manner similar to depositing thetransducer layer on the substrate. The polymer may be crosslinked andthe enzyme may be chemically modified for attachment to the polymer.Polymers useful for the enzyme layer include polyvinylalcohol,polyhydroxybutylacrylate, hydroxypropylcellulose, acrylamidederivatives, and other hydrophilic and hydrophobic polymers known tothose of skill in the art.

The protective layer is comprised of a polymer which is permeable to theanalyte while not being rapidly soluble in the sample matrix. Thepolymer can be dissolved in a solvent, which is then deposited on theenzyme layer in a similar manner. Polymers suitable for use in theprotective layer are generally those polymers described above,preferably polyhydroxyethylmethacrylate (polyHEMA). This polymer isapplied as a protective coating to prevent the enzyme from immediatelydissolving into the sample.

The fluorescent polymers can also be used as transducer coatings foroptical sensors. Traditional optical sensors for CO₂, NH₃, and otherspecies detected via a pH modulated transducer are based on theSeveringhaus model (Severinghaus, J. W.; Bradley, A. F. J. Appl.Physiol., 13: 515 (1958)) where one has a transducer layer containing apH sensitive fluorophore or chromophore, coated with a hydrophobic covermembrane material, such as a siloxane based polymer (Munkholm, C., Walt,D. R., Milanovich, F. P., Talanta, 35:109-112 (1988)). A difficultyinherent with Severinghaus sensors is their potential to fail due topinhole leaks in the cover membrane. Sensors prepared by the instantinvention will provide quantitative measurements of ammonia levels via amodulation of the microenvironment of the fluorophore. Since thesesensor microenvironments are dispersed throughout the polymer, preparingsuch a sensor requires only a single application of the membranematerial, and this single membrane configuration makes the problem ofpinhole leaks irrelevant. The sensors are not responsive to changes inthe bulk pH, indicating that the transducer microdomains are sequesteredfrom the sample. This sensor can be used in a system which measuresreflected surface fluorescence as well as in a system measuring anevanescent wave signal.

An advantage of optical sensors is their ability to resolve informationfrom different analytes via their discrete wave-bands. In this way onecould couple an ammonia sensor together with a sensor for a differentanalyte in the same membrane, but collect the readout information atseparate wavelengths. The sensor microdomains would be populated bymultiple transducers but the chemistry and signal processing would beconducted as if the sensors were in separate layers. In such amultiple-analyte sensor, the transducer for the non-ammonia analyte maybe a polyanionic dye, such as those described in U.S. Pat. No.5,506,148.

The sensor material can be used as a coating on any surface and used tomeasure creatinine in any solution, such as in blood, saliva, or urine.It could be part of products and systems used in the measurement ofcritical blood analytes, in applications used to monitor dialysis, andin all clinical point of care monitoring of creatinine. It isconceivable that this sensor material could be employed in academicresearch projects, as it could be adapted to a variety of measuringsystems, such as optical fibers.

Using the methods of this invention one can prepare extremely thinsensor films, approximately 0.5 to 5 μm thick, having a detectable levelof fluorescence. Such thin films can provide an unusually rapid responsetime and be ideal for coating planar sensors used in evanescent wavemethods of detection where one wants a fluorescent coating to be withinthe same dimensions as the propagating wave. Sensors prepared with thismethod will not be affected by pinhole leaks as the sensor material iscontinuous in the coating. These sensor films may also have a longershelflife due to their lack of an aqueous layer, which would besusceptible to dehydration.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1

The dye acridine orange was added to a 10% solution of ethyl celluloseto give 5×10⁻⁴ M. Tetrabutylammonium hydroxide was added to give a finalsolution of 0.0045 M. The solution was sonicated until mixing wascomplete. It was then spincoated onto a glass substrate and cured inambient conditions for 24 hours. A 5% solution of polyvinylalcohol indeionized water was prepared, using polyvinylalcohol of M.W.124,000-186,000. The enzyme coating was prepared by adding 1 mgcreatinine deiminase (Sigma, activity: 25-50 units per mg protein) to0.15 ml polyvinylalcohol, and then applied to the ethyl cellulose layerby spincoating. A third coating of polyHEMA (10% in methanol) wasapplied to the enzyme layer by spincoating. (FIG. 1 shows the sensorconfiguration.) The sensor was cured in ambient conditions for 24 hours.It was then tested with a commercial fluorimeter, equipped with a mountfor the sensor, using solutions of freshly prepared creatinine (2-50mg/dl) in phosphate buffer, (0.05 M, pH 7.8). FIG. 2 shows the responsecurve of the creatinine sensor, in units of fluorescence intensity, vs.concentration of creatinine in mg/dl. The signal decreased as theconcentration of creatinine increased, which is consistent with thedeprotonation of acridine orange by the production of ammonia from theenzyme catalyzed hydrolysis of creatinine.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompostions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are chemically related may be substituted for theagents described herein while the same or similar results would beachieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

What is claimed is:
 1. A creatinine sensor material comprising:a firstlayer comprising a pH-sensitive fluorophore immobilized non-aqueouslywithin a first, hydrophobic polymer, wherein the fluorophore isprotonated within the first hydrophobic polymer, can reactquantitatively with ammonia, and has a transducing moiety that isneutrally charged when deprotonated; a second layer comprisingcreatinine deiminase and a second polymer, a first side of the secondlayer contacting one side of the first layer; and a third layercomprising a third polymer, the third layer contacting a second side ofthe second layer.
 2. The sensor material of claim 1 wherein the firstlayer further comprises an onium compound.
 3. The sensor material ofclaim 2 wherein the onium compound is a quaternary ammonium compound. 4.The sensor material of claim 2 wherein the onium compound istetrabutylammonium hydroxide.
 5. The sensor material of claim 1 whereinthe fluorophore is acridine orange.
 6. The sensor material of claim 1wherein the fluorophore is a rhodamine dye.
 7. The sensor material ofclaim 1 wherein the first hydrophobic polymer is ethyl cellulose.
 8. Thesensor material of claim 1 wherein the polymer of the second layer ispolyvinyl alcohol.
 9. The sensor material of claim 1 wherein the thirdpolymer is polyhydroxyethylmethacrylate.
 10. A method for measuring theconcentration of creatinine, comprising:measuring a fluorescence of thesensor material of any of claims 1-9, exposing the sensor material to asolution comprising creatinine, measuring the fluorescence of thesensormaterial after the exposing step, determining a flourescenechange, and determining the concentration of the creatinine from thefluorescene change.
 11. A process for preparing a creatinine sensormaterial comprising:non-aqueously combining a pH-sensitive fluorophoreand a first, hydrophobic polymer to form a first mixture, wherein thefluorophore is protonated within the first, hydrophobic polymer, canreact quantitatively with ammonia, and has a transducing moiety that isneutrally charged when deprotonated; forming a first layer of the firstmixture on a substrate; combining creatinine deiminase and a secondpolymer to form a second mixture; forming a second layer of the secondmixture on the first layer; and forming a third layer of a third polymeron the second layer.
 12. The process of claim 11 wherein the combiningof the fluorophore and the first hydrophobic polymer is performed in thepresence of a solvent.
 13. The process of claim 11 wherein thefluorophore is acridine orange.
 14. The process of claim 11 wherein thefluorophore is a rhodamine dye.
 15. The process of claim 11 wherein thefirst hydrophobic polymer is ethyl cellulose.
 16. The process of claim11 wherein the polymer of the second layer is polyvinyl alcohol.
 17. Theprocess of claim 11 wherein the polymer of the third layer ispolyhydroxyethylmethacrylate.
 18. The process of claim 11 wherein thesubstrate is glass, plastic or ceramic.
 19. The process of claim 11wherein the first mixture further comprises an onium compound.
 20. Theprocess of claim 19 wherein the onium compound is a quaternary ammoniumcompound.
 21. The process of claim 19 wherein the onium compound istetrabutylammonium hydroxide.
 22. An optical sensing device formeasuring ammonia concentration in a solution comprising creatininecomprising:a first layer comprising a pH-sensitive fluorophoreimmobilized non-aqueously in a first, hydrophobic polymer, wherein thefluorophore is protonated within the first, hydrophobic polymer, canreact quantitatively with ammonia, and has a transducing moiety that isneutrally charged when deprotonated, a first side of the first layercontacting a surface of an optical component, which is transparent toincident and emissive electromagnetic waves; a second layer comprising acreatinine deiminase and a second polymer, a first side of the secondlayer contacting a second side of the first layer; a third layercomprising a third polymer, one side of the third layer contacting asecond side of the second layer; wherein the optical component isoptically connected to means for collecting radiant emission to measurethe fluorescence indicative of ammonia concentration.
 23. The device ofclaim 22 wherein the fluorophore is acridine orange.
 24. The device ofclaim 22 wherein the fluorophore is a rhodamine dye.
 25. The device ofclaim 22 wherein the first, hydrophobic polymer is ethyl cellulose. 26.The device of claim 22 wherein the polymer of the second layer ispolyvinyl alcohol.
 27. The device of claim 22 wherein the third polymeris polyhydroxyethylmethacrylate.
 28. The device of claim 22 wherein theoptical component is an optical fiber.
 29. The device of claim 22wherein the optical component is a planar waveguide.
 30. The device ofclaim 22 wherein the optical component is an evanescent wave sensor. 31.The device of claim 22 further comprising one or more sensors capable ofdetecting analytes other than ammonia.
 32. A creatinine sensor materialcomprising:a first layer consisting essentially of a pH-sensitivefluorophore immobilized non-aqueously within a first, hydrophobicpolymer, wherein the fluorophore is protonated within the first,hydrophobic polymer, can react quantitatively with ammonia, and has atransducing moiety that is neutrally charged when deprotonated; a secondlayer comprising creatinine deiminase and a second polymer, a first sideof the second layer contacting one side of the first layer; and a thirdlayer comprising a third polymer, the third layer contacting a secondside of the second layer.
 33. A creatinine sensor material comprising:afirst layer consisting essentially of a pH-sensitive fluorophore and anonium compound immobilized non-aqueously within a first, hydrophobicpolymer, wherein the fluorophore is protonated within the first,hydrophobic polymer, can react quantitatively with ammonia, and has atransducing moiety that is neutrally charged when deprotonated; a secondlayer comprising creatinine deiminase and a second polymer, a first sideof the second layer contacting one side of the first layer; and a thirdlayer comprising a third polymer, the third layer contacting a secondside of the second layer.
 34. An optical sensing device for measuringammonia concentration in a solution comprising creatinine, comprisingthe creatinine sensor material of claim 32 or 33, deposited on a surfaceof an optical component that is transparent to incident and emissiveelectromagnetic waves.